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Abstract

Background

Although national surveillance programs are in place to monitor norovirus epidemiology,
the emergence of new strains and the genetic diversity among genotypes can be challenging
for clinical laboratories. This study evaluated the analytical and clinical performance
characteristics of one real-time RT-PCR and two end-point RT-PCRs commonly used in
microbiology laboratories.

Methods

Lower limit of detection (LoD) was determined using 10-fold dilutions of noroviruses
belonging to different genotypes. The clinical performance of the real-time and end-point
RT-PCRs was assessed in parallel using nucleic acids extracted from 186 stool specimens.

Results

The real-time RT-PCR was highly sensitive and specific for the detection of norovirus
genotypes that are currently circulating in Canada. In contrast, the two end-point
RT-PCRs displayed poor analytical sensitivity or complete failure to detect certain
norovirus genotypes, which was correlated to sequence mismatches in the primer-binding
sites. In an attempt to improve norovirus detection with the end-point RT-PCRs, both
assays were processed concurrently and detection from either assay was considered
a positive result. Concurrent testing resulted in only a modest increase in clinical
sensitivity (75.0%) compared to each assay alone (62.5% and 71.9%). However, the false
positivity rate increased from 1.98% and 3.36% for the assays alone to 5.47% with
concurrent testing.

Conclusions

This study emphasizes the benefits of a real-time method and provides support for
routine surveillance to monitor norovirus epidemiology and ongoing proficiency testing
to ensure detection of circulating norovirus genotypes.

Keywords:

Background

Noroviruses are the leading cause of acute gastroenteritis, and outbreaks are common
[1,2]. Transmission occurs through the fecal-oral route and is facilitated by a low infectious
dose and environmental persistence [1,2]. Laboratory identification of norovirus can help reduce transmission through infection
control and public health interventions [2]. Since human noroviruses are uncultivable, traditional detection methods relied primarily
on electron microscopy and enzyme immunoassays, both of which lack sensitivity [2-5]. RT-PCR has markedly improved the detection of noroviruses and has become the method
of choice for clinical diagnosis [2]. However, the genetic diversity among noroviruses poses a particular challenge for
molecular assays [6-11]. Noroviruses are classified into six genogroups, three of which cause human disease
(GI, GII, and GIV) [12-14]. The two predominant genogroups, GI and GII, are further subdivided into 9 and 22
genotypes, respectively [12-14]. Strategies used to overcome norovirus diversity have included the simultaneous use
of various monoplex RT-PCRs, multiplexed RT-PCRs, RT-PCRs with degenerate primers
and probes [8-11,15-19].

The dynamic nature of its epidemiology poses further challenges for laboratory detection
of norovirus. While genotype GII.4 is responsible for the majority of outbreaks annually,
new GII.4 strains emerge every 2–3 years that replace the previously circulating pandemic
strain [14,20-25]. Norovirus GII.4-2012 Sydney has recently emerged and replaced GII.4-2009 New Orleans
worldwide [14,20-26], including the Canadian provinces of Alberta [25], British Columbia [27] and recently, Nova Scotia (unpublished data). In addition, the proportion of outbreaks
associated with non-GII.4 genotypes has increased in Canada and the US, and the predominant
non-GII.4 genotypes change over time [14,23,25,28]. With the diversity among norovirus genotypes and the dynamic nature of its epidemiology,
this study evaluated the analytical and clinical performance of a real-time RT-PCR
and two end-point RT-PCRs (EP-SR and EP-JV) commonly used for the detection of noroviruses
in clinical, food and environmental laboratories [8-11,16,17,29-36].

Results

Analytical sensitivity and specificity

Each method was specific for circulating noroviruses (Table 1) and no cross-reactions occurred with various enteric pathogens. The real-time RT-PCR
was able to detect all norovirus genotypes with high sensitivity (Table 1). EP-JV detected all GII.4 strains, but only a subset of non-GII.4 genotypes. EP-SR
only detected GII.4 strains. For GII.4 noroviruses, real-time RT-PCR was approximately
10-fold more sensitive than EP-JV, and 100-fold more sensitive than EP-SR (Table 1). Similarly, the LoD for different GII.4 strains were highly consistent for EP-JV.
For EP-SR, only minor differences in the LoD for GII.4 strains from 2004 to 2009,
but a 100-fold decrease in sensitivity was observed for GII.4-2012. Reduced sensitivity
was also observed with EP-JV for genotypes GI.6 and GII.1.

Clinical evaluation

The clinical sensitivity of the real-time RT-PCR and end-point RT-PCR assays (EP-JV
and EP-SR) was 100%, 71.9%, and 62.5%, respectively (Table 2). With concurrent testing of EP-JV and EP-SR, the clinical sensitivity was only modestly
increased to 75% compared to each assay alone, since both EP-JV and EP-SR failed to
detect 8 genotype GII.7 noroviruses (Table 3). EP-JV alone also missed a genotype GII.15 and EP-SR missed three genotype GI.6
noroviruses and one GI.3 (Table 3). With the exception of the latter, viral loads were above their LoD for all methods
and the poor sensitivity of EP-JV and EP-SR was correlated to sequence mismatches
in the primer-binding sites (Table 1 and Figure 1).

As non-specific amplifications were observed for both EP-JV and EP-SR, concurrent
testing resulted in a reduced clinical specificity (94.8%) compared to each method
alone (98.1% and 96.8%, respectively) (Table 2). The false positivity rate for concurrent testing was increased to 5.47% compared
to 1.98% and 3.36% for EP-JV and EP-SR alone. A two-year retrospective analysis revealed
that 14 outbreaks were declared by a single EP-JV and EP-SR result where weak amplifications
were noted, suggesting a possible false positivity rate of 4.8% for years 2011 and
2012 (Additional file 1: Table S2). As no differences were observed between real-time RT-PCR and the reference
methods, the real-time RT-PCR was highly specific (100%) and no false positives were
observed (Tables 2 and 3).

Discussion

Genomic diversity and evolutionary change can be challenging for detection of noroviruses
[2,37,38]. While detection of noroviruses using RT-PCR is far more accurate than antigen-based
detection methods (i.e. EIAs), not all molecular methods are created equal [2-4]. Fortunately, most reference laboratories use a real-time RT-PCR targeting the most
conserved region of the genome (ORF1-ORF2 junction) and use degenerate primers and
probes that can tolerate some sequence mismatches [9,10] (Figure 1). In this study, this same real-time RT-PCR was able to detect diverse norovirus
genotypes with analytical sensitivities consistent with values previously reported
for GII.4 (2006b) (Table 1) [6]. In contrast, EP-JV and EP-SR were far less sensitive and failed to detect certain
genotypes (Tables 1, 2 and 3). With hopes to enhance detection of circulating norovirus genotypes, EP-JV and EP-SR
were processed concurrently and detection from either assay was considered a positive
result. Concurrent testing only modestly improved clinical sensitivity (Table 2) since the majority of false negative results were common between both EP-JV and
EP-SR (Table 3). Interestingly, failure of EP-JV and EP-SR to detect certain genotypes was not attributed
to poor analytical sensitivity since the viral load in most clinical specimens far
exceeded the LoD for each assay (Tables 1 and 3). Instead, the genotypes that failed to be detected for EP-JV (GII.7, GII.15) and
EP-SR (GII.7, GI.6, and GI.3) were linked to several sequence mismatches in the primer-binding
sites (Figure 1). Of note, a large norovirus outbreak in Sweden was missed due to mismatches in the
primer-binding sites of JV12 and JV13 (same used for EP-JV), but were detected by
another primer pair [31].

It should also be noted that real-time RT-PCR and EP-JV both detected genotype GI.3
in a specimen with a very low viral load (2.47 log10 copies/ml), whereas EP-JV were unable to detect this genotype at concentrations exceeding
6.0 log10 copies/ml in the specificity analysis (Tables 1 and 3). This confounding result might be explained by genetic differences between the norovirus
GI.3 identified in the clinical evaluation and the one used for the specificity analysis.
A 12% nucleotide difference was noted between GI.6 and GI.7 genotypes that circulated
in Canada between 2009 and 2010 compared to 2012 and 2013, suggesting that diversity
among the same genotype can occur over time [24]. It is also possible that the faint amplicon detected by EP-JV was a false positive
result in a specimen that was coincidently positive by real-time RT-PCR (Table 3).

While concurrent testing marginally increased the clinical sensitivity, a reciprocal
effect was seen on the clinical specificity where the false positivity rate was higher
with concurrent testing (5.47%) than either method alone (1.98% and 3.36% for EP-JV
and EP-SR, respectively) (Table 2). Upon review of the false positive results, only faint or non-specific amplifications
were noted and likely attributed to the low annealing temperature of EP-JV (37°C)
or lack of electrophoretic resolution between primer dimmers and the small amplicon
generated with EP-SR of 123 bp. The subjectivity of gel resolution is not a problem
for a properly validated real-time RT-PCR [9,10,39].

Following a two-year retrospective analysis of EP-JV and EP-SR results, 14 of 113
outbreaks investigated in 2011 and 2012 were declared positive by a single result
where weak amplification was observed (Additional file 1: Table S2). This supports the high false positivity rate observed during the clinical
evaluation (Table 2). Previous studies have demonstrated that three specimens are ideal for the detection
of norovirus outbreaks using RT-PCR, but excessive testing can lead to reduced specificity
[5,40,41]. In this study, concurrent testing with multiple RT-PCRs was shown to increase the
rate of false positive results, which could prematurely halt outbreak investigations
caused by other enteric pathogens that might be managed differently [2].

Conclusions

Unlike the high clinical sensitivity and specificity observed with real-time RT-PCR,
this study demonstrated that end-point RT-PCRs had poor accuracy for the detection
of circulating norovirus genotypes. To monitor norovirus epidemiology, genotyping
should be considered part of routine outbreak investigations when norovirus is identified
as the etiological agent. Unlike well established networks like Calicinet and Noronet,
norovirus surveillance in Canada is in its infancy [2,14]. Ideally, sequencing would be used to encompass the regions required for genotyping
and the primer/probe-binding sites for commonly used RT-PCRs [42,43]. When sequence mismatches are identified in RT-PCR target regions or when new norovirus
variants emerges, proficiency panels should be promptly disseminated to clinical laboratories
to ensure accurate detection [6]. With the dynamic nature of norovirus epidemiology, this study highlights the importance
of routine surveillance and ongoing proficiency testing for circulating norovirus
genotypes.

Materials and methods

Specimen preparation

For the clinical evaluation, 186 stool specimens were obtained from patients with
acute gastroenteritis between March 15 and June 26, 2013. Public Health outbreak investigation
data was provided by the Department of Health and Wellness (Halifax, NS) (Additional
file 1: Table S2). Stool slurries were prepared by transferring 200 μl of stool into 500 μl
of PCR-grade water and centrifugation (10,000 × g, 10 min). The supernatants (140 μl) were subjected to a total nucleic acid (TNA)
extraction on a MagNA Pure LC instrument (Roche Diagnostics, Branchburg, NJ), as recommended
by the manufacturer. TNAs were eluted in a volume of 60 μl and 5 μl served as template
for all molecular assays (which were processed in parallel). Primers were synthesized
by Sigma Genosys (Oakville, ON) and probes by Integrated DNA Technologies (Toronto,
ON).

Discordant analysis and norovirus genotyping

Clinical sensitivity and specificity were calculated in comparison to a modified gold
standard defined as concordant positive and negative results between real-time and
end-point RT-PCRs (Table 2). Any discordant results were resolved by at the National Microbiology Laboratory
(NML) using real-time RT-PCR with the same primers/probes but in monoplex reactions
for GI and GII noroviruses (Additional file 2: Table S1). Since monoplex and duplex real-time RT-PCR targets were identical, a
second reference method was also performed at the NML using RT-PCR amplification and
sequencing of the norovirus major capsid protein regions C and D (Additional file
2: Table S1). The resulting sequence data was used for genotype assignment. Briefly,
region C and D RT-PCR reactions were performed using a One-Step RT-PCR kit (Qiagen)
in 50 μl reactions consisting of: 2 μl enzyme, 1× buffer, 400 nM dNTPs, 40 units of
RNase Inhibitor, 10 μl of template, and 500 nM each primer (except CapB1 and CapD1
used at 1 μM) (Additional file 2: Table S1). Amplification conditions were as follows: 42°C for 30 min; 95°C for 15 min;
40 cycles of 94°C for 30s, 40°C (region D) or 50°C (region C) for 30s and 72°C for
30s; and a final extension of 72°C for 10 min. Following 2% agarose gel electrophoresis,
amplicons were purified using Amicon Filter Devices (Millipore, USA) and sequencing
was carried out by the Genomics Core section of the NML using primers CapA, CapB1,
CapC, or CapD1.

Norovirus genotyping and sequence alignments

BioNumerics 5.1 software (Applied Maths, Austin, TX) was used to assemble consensus
sequence data, pairwise and global alignments, and clustering analysis. The sequences
of each region were compared to ViroNet Canada reference dataset for genotype assignment.

To compare primer/probe binding sites to the target sequences of circulating norovirus
genotypes (Figure 1), sequence data was retrieved from the Genbank database on the NCBI website (http://www.ncbi.nlm.nih.govwebcite) and pairwise sequence alignments were performed using the Basic Local Alignment
Search Tool (BLAST) function.

Statistical analysis

Chi-square and two-tailed Fisher's exact tests were used to compare proportions in
2-by-2 contingency tables. Binomial 95% confidence intervals and kappa statistics
for each parameter were calculated by the "constant chi-square boundaries" method
[45] using StatsPlus version 5.8.4.3 (AnalystSoft, Inc.).

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

BR and JL carried out the specimen processing and real-time and end-point RT-PCRs.
JP performed the outbreak investigations. Discrepant analysis was performed by EG
and TB. OM cloned the plasmids used for viral load determination. Specificity panels
were prepared by XP. TB, TH, and JL were involved in the coordination and design of
the study. All authors helped draft the manuscript and the final version was approved
by all authors.

Acknowledgements

We would like to thank the Nova Scotia Provincial Public Health Laboratory Network
(PPHLN) for providing funding for the project and members of the Division of Microbiology
at CDHA for their ongoing support. We are also indebted to the Genomic Core Section
at the NML.